Papillomaviruses (PV) are non-enveloped DNA viruses that induce hyperproliferative lesions of the epithelia. The papillomaviruses are widespread in nature and have been recognized in higher vertebrates. Viruses have been characterized, amongst others, from humans, cattle, rabbits, horses, and dogs. The first papillomavirus was described in 1933 as cottontail rabbit papillomavirus (CRPV). Since then, the cottontail rabbit as well as bovine papillomavirus type 1 (BPV-1) have served as experimental prototypes for studies on papillomaviruses. Most animal papillomaviruses are associated with purely epithelial proliferative lesions, and most lesions in animals are cutaneous. In the human there are more than 75 types of papillomavirus (HPV) that have been identified and they have been catalogued by site of infection: cutaneous epithelium and mucosal epithelium (oral and genital mucosa). The cutaneous-related diseases include flat warts, plantar warts, etc. The mucosal-related diseases include laryngeal papillomas and anogenital diseases comprising cervical carcinomas (Fields, 1996, Virology, 3rd ed. Lippincott--Raven Pub., Philadelphia, N.Y.).
There are more than 25 HPV types that are implicated in anogenital diseases, these are grouped into "low risk" and "high risk" types. The low risk types include HPV type 6, type 11 and type 13 and induce mostly benign lesions such as condyloma acuminata (genital warts) and low grade squamous intraepithelial lesions (SIL). In the United States there are 5 million people with genital warts of which 90% is attributed to HPV-6 and HPV-11. About 90% of SIL is also caused by low risk types 6 and 11. The other 10% of SIL is caused by high risk HPVs.
The high risk types are associated with high grade SIL and cervical cancer and include most frequently HPV types 16, 18, 31, 33, 35, 45, 52, and 58. The progression from low-grade SIL to high-grade SIL is much more frequent for lesions that contain high risk HPV-16 and 18 as compared to those that contain low risk HPV types. In addition, only four HPV types are detected frequently in cervical cancer (types 16, 18, 31 and 45). About 500,000 new cases of invasive cancer of the cervix are diagnosed annually worldwide (Fields, 1996, supra).
Treatments for genital warts include physical removal such as cryotherapy, CO.sub.2 laser, electrosurgery, or surgical excision Cytotoxic agents may also be used such as trichloroacetic acid (TCA), podophyllin or podofilox. Immunomodulatory agents are also available such as Interferon or Imiquimod. These treatments are not completely effective in eliminating all viral particles and there is either a high cost incurred or uncomfortable side effects related thereto. In fact, there are currently no effective antiviral treatments for HPV infection since with all current therapies recurrent warts are common (Beutner & Ferenczy, 1997, Amer. J. Med., 102(5A), 28-37).
The ineffectiveness of the current methods to treat HPV infections has demonstrated the need to identify new means to control or eliminate such infections. In recent years, efforts have been directed towards finding antiviral compounds, and especially compounds capable of interfering with viral replication at the onset of infection (Hughes, 1993, Nucleic Acids Res. 21:5817-5823). To that end, it has therefore become important to study the genetics of HPVs in order to identify potential chemotherapeutic targets to contain and possibly eliminate any diseases caused by HPV infections at the onset of infection. It is equally important to identify a measurable viral activity that demonstrates specificity and reliability to be used as an indicator in assessing the effectiveness of the potential chemotherapeutic agents against PVs.
The life cycle of PV is closely couple d to keratinocyte differentiation. Infection is believed to occur at a site of tissue disruption in the basal epithelium. As the infected cells undergo progressive differentiation the cellular machinery is maintained allowing viral gene expression to increase, with eventual late gene expression and virion assembly in terminally differentiated keratinocytes and the release of viral particles (Fields, supra).
The coding strands for each of the papillomavirus contains approximately ten designated translational open reading frames (ORFs) that have been classified as either early ORFs or late ORFs based on their location in the genome. E1 to E8 are expressed early in the viral replication cycle, and two late genes (L1 and L2) represent the major and minor capside proteins respectively. The E1 and E2 gene products function in viral DNA replication, whereas E5, E6 and E7 are expressed in connection with host cell proliferation. The L1 and L2 are involved in virion structure. The functions of E3, E4 and E8 gene products is uncertain at present.
Studies of HPV have shown that proteins E1 and E2 are both essential and sufficient for viral DNA replication in vitro (Kuo et al., 1994, J. Biol. Chem. 30: 24058-24065). This requirement is similar to that of bovine papillomavirus type 1 (BPV-1). Indeed, there is a high degree of similarity between E1 and E2 proteins and the ori-rsequences of all papillomaviruses (PV) regardless of the viral species and type (Kuo et al., 1994, supra). Of note, E1 is the most highly conserved protein in PV and its enzymatic activity is presumed to be similar for all PV types (Jenkins, 1996, J. Gen. Virol., 77:1805-1809).
Evidence emanating from studies of BPV-1 have shown that E1 possesses ATPase and helicase activities that are required in the initiation of viral DNA replication (Seo et al., 1993a, Proc. Natl. Acad. Sci. USA 90:702-706; Yang et al., 1993, Proc. Natl. Acad. Sci. 90:5086-5090; and MacPherson et al., 1994, 204:403-408).
The E2 protein is a transcriptional activator that binds to E1 protein and forms a complex that binds specifically to the ori sequence (see FIG. 1) (Mohr et al., 1990, Science 250:1694-1699). It is believed that E2 enhances binding of E1 to the BPV origin of replication (Seo et al., 1993b, Proc. Natl. Acad. Sci., 90:2865-2869). In HPV, Lui et al. suggested t hat E2 stabilizes E1 binding to the ori (1995, J. Biol. Chem., 270(45):27283-27291).
The helicase activity of the E1 proteins of papillomavirus therefore constitute a good molecular target to design chemical entities capable of inhibiting viral replication. Such objective requires that the E1 protein be extracted and purified to an extent where its helicase activity can be measured reliably and reproducibly. Such isolation of E1 helicase has however remained elusive or at best unreliable, especially on a scale sufficient to establish an assay to screen for such inhibitors.
Seo et al. (1993a, supra) disclose the extraction and purification of BPV-E1 from a baculovirus expression system with the step consisting of the use of PEG and 1 M NaCl in the nuclear extraction buffer. They obtained BPV-E1 preparation about 90% pure. However, we have not found it possible to obtain pure HPV-11 E1 by this procedure, and in any case the procedure is not suitable to the large scale required to purify E1 for high-throughput screening.
The two BPV-1 genes encoding E1 and E2 proteins have been cloned into a Baculovirus expression system and the proteins substantially purified (U.S. Pat. No. 5,464,936). U.S. Pat. No. '936 discloses a purification process for E1 consisting of a nuclear extraction in a hypertonic buffer (containing 300 mM NaCl) followed by 3 sequential chromatographic separations. The disclosure, however, does not demonstrate the purity and specific activity of the resulting E1 helicase. The absence of affinity chromatography purification step leads to the presence of contaminating nucleases that prevent accurate measurement of the E1 helicase activity. In addition, even if such a process would in fact yield E1 helicase of sufficient purity to assess the helicase activity, it is believed that it would be inapplicable to a high-yield, large-scale process for HTS purposes.
An extraction process wherein nuclei were suspended in lysis buffer containing 300 mM NaCl followed by further purification has been described (Bream et al., 1993, J. Virol., 2655). However, the authors were unable to detect helicase activity from these crude preparations of E1. Further attempts to isolate HPVs E1 protein cloned into different expression systems, having demonstrable and specific helicase activity, have failed (Jenkins et al., 1996, supra).
Kuo et al., 1994, supra discloses a purification procedure (using 420 mM salt during the nuclear extraction) but does not discuss the scale on which the procedure was carried out or the total yield of protein.
It has been hypothesized that the conformation of the E1 protein and its hydrophobicity cause the protein to be "sticky" and to form aggregates thus making it difficult to extract and purify. In addition, difficulties in establishing enzymatic activities that are specific and free of cellular contaminants have generally been encountered. For example, viral helicase and/or ATPase activity may not be distinguishable from cellular helicase and/or ATPase contaminants present in the host cell used to express the E1 gene. In addition, very low levels of nucleases will destroy the substrate rendering any assessment impossible.
One common denominator in the various purification processes outlined above lies in the presence of high concentrations of salt (hypertonic conditions) during the nuclear extraction step. Indeed, according to conventional wisdom, it is believed that nucleic acid-binding proteins may be solubilized in high concentrations of salt and thereby separated from nucleic acids. At present, the prior art has not revealed satisfactory processes for the purification of E1.
There thus remains a need to isolate a demonstrable and reproducible viral helicase activity that can be used as an indicator of the inhibitory effect of antiviral chemotherapeutic agents. More particularly, there remains a need to provide a method of preparing a PV E1 preparation displaying a high helicase activity.
There also remains a need to obtain a preparation of human papillomavirus E1 protein which displays a helicase activity sufficient for the purposes of a screening assay, particularly, a high throughput screening assay.
Since E1 structure/function is highly conserved amongst different papillomaviruses and amongst subtypes, it is assumed that the BPV and CRPV E1 proteins can be extracted and purified by the procedure of the invention. Therefore, there remains a need for a method for the isolation/purification of E1 protein from several species of papillomavirus, including, but not limited to bovine papillomavirus (BPV), cottontail rabbit papillomavirus (CRPV) and human papillomavirus (HPV). There also remains a need to isolate and purify the E1 protein from different subtypes of HPV, including but not limited to, HPV-6, 11, 16, 18, 31, 33, 35, 45, 52, and 58.
Before the present invention, E1 protein preparations, including human E1 preparations, did not demonstrate reproducible helicase activity. The deficiency in the prior art created a road block in being able to screen a large collection of antiviral agents capable of inhibiting papilloma viral DNA replication. This deficiency is overcome by the present invention which is capable of providing the means to design a HTS for the screening for such agents. The Applicant has now found a reliable and reproducible purification process for the preparation of E1 having helicase activity. The resulting E1 preparation is free from degradation products and amenable to large scale production of E1.
The present description refers to a number of documents, the content of which is herein incorporated by reference.